Chemokine receptor able to bind to MCP-1, MIP-1α and/or RANTES. Its uses

ABSTRACT

A chemokine receptor binds to MCP-1, MIP-1α and/or RANTES. It can be used in screening for agents which act as antagonists to MCP-1, MIP-1α and/or RANTES. Such agents may be useful in treating various disorders, including allergies, atheromas and diseases mediated by viruses. They may also be useful in preventing graft rejection and in protecting stem cells from potentially damaging effects of chemotherapy.

The present invention relates to chemokine receptors.

Chemokines are a growing family of chemotactic cytokines, which havebeen implicated to play a role in the recruitment and activation ofcells (Oppenheim, J. J. et al., Ann Rev Immunol., 9 617-48, (1991),Schall, T. J., Cytokine, 3 165-183, (1991)). They are primarilyresponsible for the activation and recruitment of leukocytes, but notexclusively so. Further analysis of this superfamily of proteins hasshown that it can be divided up into two further subfamilies ofproteins. These have been termed CXC or α-chemokines, and the CC orβ-chemokines based on the spacings of two conserved cysteine residuesnear to the amino terminus of the proteins.

To date two receptors have been identified for the CC chemokine family.The first, which is receptor primarily for MIP-1α (Macrophageinflammatory polypeptide-1α) and RANTES (Raised on Activation, NormalT-cell derived and Secreted) has been described previously (Gao, J. L.et al., J. Exp. Med., 177 1421-7 (1993), Neote, K. et al., Cell 72415-25 (1993)). The second CC-chemokine receptor which has been recentlydescribed is for MCP-1 (monocyte chemotractant protein-1) Charo I. etal., Proc. Natl. Acad. Sci. USA 91 2752-2756 (1994). More recently,another receptor US28, expressed by the human cytomegalovirus, has beenshown to be a receptor for RANTES, MIP-1α, and MCP-1 (Gao, J. L. andMurphy P. M., J. Biol. Chem. 269, 28539-28542 (1994)). All threereceptors are of the seven transmembrane alpha helical segment type, andare expressed into the membranes of cells.

However there remains a need to identify hitherto undisclosed chemokinereceptors and to characterise them in order to develop a more completepicture of the structure and function of chemokine receptors.

According to the present invention there is provided a chemokinereceptor having the amino acid sequence shown in FIG. 3.

This receptor is preferably capable of binding MCP-1, MIP-1α and RANTES.It may be important in basophil and T-cell function.

It can be used to screen for pharmaceutically active agents. The presentinvention therefore includes within its scope such agents (which may ormay not be proteins). They may be provided in a pharmaceuticalcomposition together with a pharmaceutically acceptable carrier.

Such a composition is within the scope of the present invention. It maybe prepared by admixing the carrier with the pharmaceutically activeagent under sterile conditions. The pharmaceutical composition may beprovided in unit dosage form. It may be present as part of a kitincluding instructions for use.

The pharmaceutical composition may be adapted for administration by anyappropriate route, for example by the oral (including buccal orsublingual), rectal, nasal, topical (including buccal, sublingual ortransdermal), vaginal or parenteral (including subcutaneous,intramuscular, intravenous or intradermal) route.

A receptor of the present invention can be used to screen for agentsuseful in treating allergies e.g. asthma, atopic dermatitis, rhinitis,hay fever, eczema or food allergies. It may also be useful in screeningfor agents useful in treating conjunctivitis. MCP-1, MIP-1α and RANTESall bind to the receptor of the present invention and are capable ofcausing histamine release from basophils. An agent which blocks thisbinding may thereby prevent or reduce the release of histamine frombasophils (i.e. act antagonistically to MCP-1, MIP-1α or RANTES). Suchagents may be variants of MCP-1, MIP-1α or RANTES (in which one or moreamino acids are deleted, substituted or inserted relative to MCP-1,MIP-1α or RANTES), although this is not essential.

It may also be involved in the activation of T-lymphocytes, a commoncharacteristic of immune and other inflammatory states.

The binding of agents to the receptor of the present invention can beassayed by suitable techniques.

For example, electrophysiological techniques may be used. In one suchtechnique, a Xenopus oocyte, for example, can be used to express areceptor of the present invention. The receptor can be expressed on theoocyte membrane following micro-injection into the oocyte of RNA codingfor said receptor.

When a ligand binds to the receptor, it can cause a release of calciumions either from intracellular stores, or from extracellular sources.These calcium fluxes then cause a chloride current across the cellmembrane which can be measured electrophysiologically.

Such currents are discussed in Wahlestedt, C., Ann. N.Y. Acad. Sci. 632116-22 (1991) and Boton, R et al., J. Physiol. (London) 408 511-534(1989), for example.

As an alternative to using electrophysiological techniques or othertechniques which rely upon a biological response to receptor binding,more direct assays of binding can be used. Thus ligands could belabelled with a detectable label, allowed to bind to a receptor, and thelabel could then be detected. Suitable labels which could be usedinclude radiolabels, fluorescent labels, enzymes which can cause adetectable change, etc.

The receptor of the present invention may also be used to screen foragents suitable for treating atheromas. In this regard it should benoted that MCP-1 is a key recruiter of monocytes to atheroscleroticplagues. The receptor can be used to screen for agents which prevent orreduce such recruitment (act antagonistically to MCP-1α). Such agentsmay be variants of MCP-1 itself (wherein one or more amino acids aredeleted, substituted or inserted relative to MCP-1), although this isnot essential.

A further use of the receptor of the present invention is to screen foragents which cause inhibition of stem cell proliferation, in other wordsto screen for agonists of MIP-1α. MIP-1α has been shown (Graham, G. J.et al., Nature 344 442- (1990)) to inhibit proliferation of hemopoeticstem cell proliferation. As such, receptor agonists could be used toprevent stem cell proliferation during chemotherapy, which wouldtherefore protect the stem cells from the potentially damaging effectsof such chemotherapy.

MIP-1α is known to be a stem cell proliferation inhibitor and agentswhich are also stem cell proliferation inhibitors can be screened usingthe receptor of the present invention. Such agents may be variants ofMIP-1α itself (wherein one or more amino acids are deleted, substitutedor inserted relative to MIP-1α), although this is not essential.

Another use of the receptor of the present invention is in screening foragents useful in reducing the likelihood of transplant rejection or inincreasing the length of time before rejection occurs. High levels ofRANTES are sometimes found in renal grafts and may be associated withthe rejection of such grafts. Agents which prevent or reduce the bindingof RANTES to the receptor of the present invention may therefore beuseful in transplantation by acting antagonistically to RANTES. Suchagents may be variants of RANTES itself (wherein one or more amino acidsare deleted, substituted or inserted relative to RANTES), although thisis not essential.

A further use of the present invention is in screening for substancesuseful in treating diseases mediated by viruses. Thus it may be used asa screen for antiviral agents.

One example of this is in screening for agents useful in treating AIDS.MIP-1α and RANTES levels have been suggested as being at least partiallyresponsible for certain AIDS patients surviving longer than others.Since a receptor of the present invention may bind to MIP-1α and/orRANTES, it can be used for screening for other agents which could beuseful in treating AIDS.

It is also notable that Human Cytomegalovirus and Herpes viruses havechemokine receptors. The present invention could be used to screen foragents useful in treating diseases mediated by such viruses.

It should be noted that the present invention is not limited to thereceptor having the amino acids sequence shown in FIG. 3 but that itcovers variants (allelic and non-allelic variants) having one or moreamino acid deletions, insertions or substitutions relative to saidsequence, provided that said variants are capable of binding to at leastone of the chemokines: RANTES, MIP-1α and MCP-1. (Desirably, however,the receptors are capable of binding to all of these chemokines).Binding may be determined by monitoring the response of cells inelectrophysiological assay using oocytes, as already described.

For example, it will be appreciated by the skilled person that variousamino acids may often be substituted for other amino acids which havesimilar properties without substantially altering or adversely affectingcertain properties of a protein. Thus the amino acids glycine, valine,leucine or isoleucine can often be substituted for one another (aminoacids having aliphatic hydroxyl side chains). Other amino acids whichcan often be substituted for one another include: phenylalanine,tyrosine and tryptophan (amino acids having aromatic side chains);lysine, arginine and histidine (amino acids having basic side chains);aspartate and glutamate (amino acids having acidic side chains);asparagine and glutamine (amino acids having amide side chains) andcysteine and methionine (amino acids having sulphur containing sidechains). Thus the present invention includes within its scope variantsof the receptor shown in FIG. 3 which includes one or more suchsubstitutions.

It is however preferred that variants of the receptor having the aminoacid sequence shown in FIG. 3 have substantial amino acid identity withsaid amino acid sequence. The degree of amino acid identity can becalculated using a program such as "bestfit" (Smith and Waterman,Advances in Applied Mathematics, 482-489 (1981)) to find the bestsegment of similarity between the two sequences. The alignment is basedon maximising the score achieved using a matrix of amino acidsimilarities, such as that described by Schwarz and Dayhof (1979) Atlasof Protein Sequence and Structure, Dayhof, M. O., Ed pp 353-358.

Preferably however the degree of sequence identity is at least 50% or atleast 60%, and more preferably it is above 75%. Sequence identities ofat least 80%, e.g. at least 90% or at least 95%, are most preferred.

The receptor or variant thereof may include an N-terminal methionine.Such methionines are sometimes incorporated during translation and notsubsequently removed.

The receptor or variant may be covalently linked to another moiety (e.g.a protein). Thus fusion proteins may be formed. These are well known inthe art and may be used to assist in identification or purification orto otherwise alter the properties of the receptor of a variant thereof(e.g. to alter its stability and/or is binding properties).

Truncated variants of the receptor having the amino acid sequence shownin FIG. 3 may also be provided since one or more amino acids may bedeleted from said sequence, whilst retaining binding to MIP-1α, RANTESand/or MCP-1. These may be N-terminal deletions, C-terminal deletions ormay occur within said sequence.

The receptor or variant (of whatever nature) may be provided insubstantially pure form. It may be isolated from other proteins and maybe isolated from a cell membrane. It may be in glycosylated orunglycosylated form (depending upon the expression system used). Areceptor or variant thereof of the present invention may be provided byany appropriate technique.

Gene cloning techniques are preferably used. Such techniques aredisclosed, for example, in J. Sambrook et al., Molecular Cloning 2ndEdition, Cold Spring Harbor Laboratory Press (1989).

Alternatively, chemical synthesis may be used (although this is lesspreferred). For example, short synthetic peptides may be prepared andthen linked together to provide a substance of the present invention.Such peptides can be prepared by techniques known to those skilled inthe art. Thus one end of a molecule can be immobilised and desired aminoacid residue can be added sequentially. Protective groups can be used toavoid undesired side-reactions and may then be removed.

Variants of the receptor of the present invention together with thereceptor itself are referred to below as substances of the presentinvention.

Such substances can be used in raising or selecting antibodies. Thepresent invention therefore includes antibodies which bind to asubstance of the present invention. Preferred antibodies bindspecifically to substances of the present invention so that they can beused to purify such substances. The antibodies may be monoclonal orpolyclonal.

Polyclonal antibodies can be raised by stimulating their production in asuitable animal host (e.g. a mouse, rat, guinea pig, rabbit, sheep, goator monkey) when the substance of the present invention is injected intothe animal. If necessary an adjuvant may be administered together withthe substance of the present invention. The antibodies can then bepurified by virtue of their binding to a substance of the presentinvention.

Monoclonal antibodies can be produced from hybridomas. These can beformed by fusing myeloma cells and spleen cells which produce thedesired antibody in order to form an immortal cell line. This is thewell known Kohler & Milstein technique (Nature 256 52-55 (1975)).

Techniques for producing monoclonal and polyclonal antibodies which bindto a particular protein are now well developed in the art. They arediscussed in standard immunology textbooks, for example in Roitt et al,Immunology second edition (1989), Churchill Livingstone, London.

In addition to whole antibodies, the present invention includesderivatives thereof which are capable of binding to substances of thepresent invention.

Thus the present invention includes antibody fragments and syntheticconstructs. Examples of antibody fragments and synthetic constructs aregiven by Dougall et al in Tibtech 12 372-379 (September 1994).

Antibody fragments include, for example, Fab, F(ab')₂ and Fv fragments(see Roitt et al [supra]).

Fv fragments can be modified to produce a synthetic construct known as asingle chain Fv (scFv) molecule. This includes a peptide linkercovalently joining V_(h) and V_(l) regions which contribute to thestability of the molecule.

Other synthetic constructs include CDR peptides. These are syntheticpeptides comprising antigen binding determinants. Peptide mimetics mayalso be used. These molecules are usually conformationally restrictedorganic rings which mimic the structure of a CDR loop and which includeantigen-interactive side chains.

Synthetic constructs include chimaeric molecules. Thus, for example,humanised (or primatised) antibodies or derivatives thereof are withinthe scope of the present invention. An example of a humanised antibodyis an antibody having human framework regions, but rodent hypervariableregions.

Synthetic constructs also include molecules comprising a covalentlylinked moiety which provides the molecule with some desirable propertyin addition to antigen binding. For example the moiety may be a label(e.g. a fluorescent or radioactive label) or a pharmaceutically activeagent.

The antibodies or derivatives thereof of the present invention have awide variety of uses. They can be used in purification and/oridentification of the substances of the present invention. Thus they maybe used in diagnosis.

They can be provided in the form of a kit for screening for thesubstances of the present invention.

The present invention also includes within its scope nucleic acidmolecules coding for substances of the present invention (i.e. for theaforesaid receptor or variants thereof). The nucleic acid molecules maybe RNA or DNA and may be provided in isolated or recombinant form.

Nucleic acid molecules of the present invention may be provided by anyappropriate technique.

Gene cloning techniques are preferred (see Sambrook et al, supra).Variants of a given nucleic acid sequence can be prepared by mutagenesistechniques (e.g. site directed mutagenesis).

Chemical synthesis techniques can alternatively be used, but are lesspreferred.

Vectors may be used to incorporate the nucleic acid molecules of thepresent invention. The vectors may be eukaryotic or prokaryotic vectorsand may be incorporated into appropriate host cells or into non-cellularexpression systems.

Nucleic acid molecules which are complementary to the aforesaid nucleicacid molecules are also within the scope of the present invention. Theseare sometimes referred to as "antisense molecules". They can hybridiseto complementary nucleic acid molecules and may thereby prevent orreduce the expression of a gene product. Thus they can be used to altergene expression.

The use of such molecules is useful in studying gene function andregulation. Appropriate labelling and hybridisation techniques can beuseful to identify the location of coding regions.

The present invention also includes within its scope nucleic acids whichcan be used as probes for chemokine receptors. Preferred probes canhybridise specifically to a nucleic acid coding for the protein havingthe amino acid sequences given in FIG. 3, or for variants thereof, asdescribed above. Such probes can be of any suitable length, but wouldtypically be above 15 nucleotides long and may be at least 100nucleotides long.

Desirably probes will hybridise to a target sequence under stringenthybridisation conditions. An example of stringent hybridisationconditions is a temperature of 35°-65° C. and a salt concentration ofabout 0.9 molar. Other stringent hybridisation conditions may be usedand the salt concentration need not be as high as 0.9 molar.

The nucleic acid sequences given in FIGS. 1 and 3 herein or fragmentsthereof can be used as probes or primers or to prepare probes orprimers.

The primers may be used to amplify nucleic acid sequences e.g. by usingPCR or other amplification techniques. The probes may be used indiagnosis or in purification.

The present invention will now be explained by way of example only, withreference to the accompanying drawings, wherein:

FIG. 1 shows a cDNA sequence (SEQ ID NO:5) and a deduced amino acidsequence (SEQ ID NO:6) of a clone designated "TM(2-7)5.5", which wasused to probe a human spleen λGT11 cDNA library.

FIG. 2 shows various primers (SEQ ID NOS 7-18, respectively) which wereused in sequencing a clone isolated from the library referred to inrespect of FIG. 1 above by using the TM(2-7)5.5 DNA as a probe.

FIG. 3 shows the cDNA sequence (SEQ ID NO:19) and the deduced amino acidsequence (SEQ ID NO:20) in respect of the clone referred to in respectof FIG. 2 above, the clone being designated "K5.5".

FIGS. 4 to 6 show Northern Blot analyses prepared using TM(2-7)5.5 DNAin various hybridisation studies. In these figures the following scoringsystem is used:

+++ Very strong positive signal visible after four hours' exposure ofthe autoradiograph.

++ Clear positive signal visible after four hours' exposure of theautoradiograph.

+ Signal not visible after four hours' exposure of the autoradiograph,but clear after 24 hours.

+/- Weak positive signal only visible after 24 hours' exposure.

- No signal.

Probes were used at a specific activity of 10⁶ cpm/ml hybridisationsolution.

FIG. 7 shows on agarose gels an analysis of K5.5 receptor mRNAexpression products from leukocytes and some human cell lines, RNAhaving been amplified using reverse transcriptase PCR.

FIG. 8A shows an analysis of the current induced in voltage clampedXenopus oocytes, into which K5.5 cRNA had been micro-injected, onstimulation with various chemokine ligands.

FIG. 8B shows an analysis similar to that performed in respect of FIG.8A but using different chemokines (apart from MIP-1α, which is shown inboth figures for comparison). The present inventors were unable toobtain any data showing that IL-8 binds to CC-CKR3 molecules. Preferredreceptors within the scope of the present invention do not bind to IL-8.

FIG. 9 shows the results of a binding assay using [¹²⁵ I] MIP-1α and[¹²⁵ I] RANTES to bind to human and murine CC-CKR-3 molecules.

EXAMPLES

An alignment of the amino acid sequences of IL-8 receptor A and B and ofthe C-C CKR-1(MIP-1α/RANTES receptor) indicated that a region betweenproposed transmembrane domains 3 and 4 contains the conserved amino acidsequence R Y L A I V H A (SEQ ID NO:1.

A second conserved amino acid sequence occurs in the proposed 7thtransmembrane domain in these three receptors as well as in twonon-chemokine chemotactic peptide receptors for fMLP(formyl-methionine-leucine-phenylalanine) and C5a as follows (SEQ IDNO:2):

C L(or V,I) N P I(or L,M,V) I(or L) Y A(or V) F(or V)

Degenerate oligonucleotides were prepared containing the majority ofpossible codons which could be translated to give the above-mentionedamino acid sequences.

These oligonucleotides had the sequences (SEQ ID NOS 3 and 4,respectively):

a) sense 5' GIT AYY TIG CIA THG TIC AYG C or

b) antisense 5' AMI RCR TAI ADI AII GGR TTI AIR C using the IUB/GCGcodes, wherein

I=inosine which can substitute for A, T, G, or C

Y=C or T

H=A, C or T

M=A or C

R=A or G

D=A, G or T

The oligonucleotides were then used to clone a human CC chemokinereceptor using the procedure set out below.

(a) Cloning of a Sequence Designated Human K5.5 (CC-CKR-3)*

Total RNA was isolated from 1×10⁸ KU812 cells by the method ofChomczynski and Sacchi, (1987) (Single-step method of RNA isolation byacid guanidinium thiocyanate-phenol-chloroform extraction, Anal.Biochem. 162 156-159). These cells were from a human basophilic KU812cell line which was a gift of Dr K Kishi, Niigata, Japan.

PolyA+ mRNA was subsequently isolated by oligodT cellulosechromatography using a polyA quik mRNA purification kit (Stratagene).Single-stranded cDNA was prepared from 1 μg of polyA+ mRNA in a 50 μlreaction containing 1 μg oligodT₁₂₋₁₈, 4 mM methyl mercuric hydroxide, 1mM dNTPs, 50 mM Tris-HCl pH 8.3 buffer, 50 mM KCl, 8 mM MgCl₂, 10 unitsRNAsin and 100 units of AMV reverse transcriptase-XL (Life SciencesInc.) for 60 min at 42° C. 5 μl aliquots of the reaction mixture werethen subjected to 40 cycles of PCR (95° C., 2 min; 37° C., 2 min and 72°C., 2 min) in 10 mM Tris-HCl pH 8.3 buffer, 50 mM KCl, 1.5 mM MgCl₂, 0.2mM dNTPS and 2.5 units of Amplitaq™ (Perkin Elmer Cetus) using 3 μM ofeach degenerate oligonucleotide primer (SEQ ID NOS 3 and 4,respectively) (sense 5' GIT AYY TIG CIA THG TIC AYG C and antisense 5'AMI RCR TAI ADI AII GGR TTI AIR C) in a Techne PHC-2 thermal cycler.

PCR reaction products were visualized on 1% agarose gels containing 0.5μg/ml ethidium bromide. Reaction products migrating at the predictedsize (500-550 bp) were gel purified by standard methods (Sambrook J. etal., 1989 in Molecular Cloning: A Laboratory Manual. Cold Spring HarborLaboratory Press. Cold Spring Harbor, N.Y.). Gel purified DNA was thenrendered blunt-ended by sequential treatment with T4 polynucleotidekinase (New England Biolabs) according to the manufacturer'sinstructions, in a total volume of 50 μl for 1 h at 37° C. After thistime, 2.5 μl of 2.5 mM dNTPs and 1 μl of E. coli DNA polymerase I Klenowfragment (New England Biolabs) were added and the incubation continuedfor a further 30 min at 37° C. The reaction mixture was then heatinactivated at 70° C. for 30 min and then extracted once with Tris-HClpH 8.0 saturated phenol/chloroform (1:1 v/v). DNA was precipitated byaddition of 10 μl 3M sodium acetate pH 5.5, 1 μl glycogen (20 mg/ml)(Boehringer) and 250 μl ethanol at -20° C. The DNA was recovered bycentrifugation at 10 000×g for 20 min at 4° C. and washed with 70%ethanol. The final pellet was resuspended in sterile water at aconcentration of 10 ng/μl.

A pBluescript II SK- cloning vector (Stratagene) was prepared asfollows: 20 μg of CsCl gradient purified plasmid was digested in areaction volume of 100 μl for 2 h at 37° C. with 200 units of Eco RV orEco RI (New England Biolabs) according to the manufacturer'sinstructions. After 2 h, the digested vector was treated with 10 μl ofcalf intestinal alkaline phosphatase (20 units/ml) (Boehringer) for afurther 30 min at 37° C.

The reaction mixture was inactivated by heating at 68° C. for 15 min andthen extracted once with Tris-HCl pH 8.0 saturated phenol/chloroform(1:1 v/v). Plasmid DNA was precipitated by addition of 10 μl 3M sodiumacetate pH 5.5 and 250 μl ethanol at -20° C. The DNA was recovered bycentrifugation at 10 000×g for 20 min at 4° C., washed with 70% ethanol.The final pellet was resuspended in sterile water at a concentration of50 ng/ml.

Blunt-ended PCR product (10 ng) was ligated to 5 ng of Eco RV digested,alkaline phosphatase treated pBluescript II SK- plasmid cloning vectorin a 20 μl volume using 2 μl of T4 DNA ligase (400 000 units/ml) (NewEngland Biolabs) for at least 16 h at 15° C. Ligation products werediluted to 100 μl with 1×TE (10 mM Tris-HCl pH 8.0/1 mM EDTA) andphenol/chloroform extracted as described previously. Ligation productswere precipitated by the addition of 10 μl 3M sodium acetate pH 5.5, 1μl glycogen (20 mg/ml) and 250 μl ethanol for 15 min at -70° C. DNA wasrecovered by centrifugation as described above and resuspended in 10 μlof sterile water. Five μl of resuspended ligation products were thenelectroporated into electrocompetent E. coli strain XL-1 blue (recA1,endA1, gyrA96, thi-1, hsdR17, supE44, relA1, lac, {F' proAB, lacIqZDM15,Tn10 (tet^(r))] (40 μl) using a Bio Rad Gene pulser according to themanufacturer's instructions. Following electroporation, 1 ml of LBmedium was added and cells were grown at 37° C. for 1 h. After thistime, 100 μl aliquots of the culture medium were plated on LB platescontaining 100 μg/ml of ampicillin and grown up for 16 h at 37° C.Individual bacterial colonies were then picked into 5 ml of LB mediumcontaining 100 μg/ml of ampicillin and grown overnight at 37° C. Smallscale plasmid DNA preparations (mini-preps) were then made from 3 ml ofeach culture using a Wizard™ mini-prep DNA purification system (Promega)according to the manufacturer's instructions. Three μl aliquots ofmini-prep DNA was then digested with restriction enzymes Hind III andEco RI (both from New England Biolabs) according to the manufacturer'sinstructions in a reaction volume of 15 μl. Reaction products wereanalysed on 1% agarose gels containing 0.5 μg/ml ethidium bromide.Mini-prep DNAs which yielded an insert size of approximately 500-550 bpwere then subjected to DNA sequence analysis using T3 and T7 primers andSequenase (USB) according to the manufacturer's instructions.

A comparison of the sequences obtained against the GenBank/EMBL/DDBJdatabases revealed that 10/23 sequences analysed showed 60% identity atthe DNA level to the human C-C CKR-1 (MIP-1α/RANTES receptor) (Neote etal., Molecular cloning, functional expression and signallingcharacteristics of a C-C chemokine receptor, Cell 72 415-425 (1993)).The sequence of one of the clones designated TM(2-7)5.5 (shortened toK5.5) is shown in FIG. 1.

CsCl gradient-purified plasmid DNA was prepared for clone K5.5 bystandard methods. 20 μg of plasmid DNA was digested at 37° C. withrestriction enzymes Hind III and Eco RI according to the manufacturer'sinstructions (New England Biolabs). Digestion products were analysed on1% agarose gels containing 0.5 μg/ml ethidium bromide. The 514 bp insertDNA corresponding to the sequenced PCR product was gel purified asdescribed previously. One hundred ng of the 514 bp insert was labelledwith ³² P-dCTP (Amersham International) using a random-primedDNA-labelling kit (Boehringer) according to the manufacturer'sinstructions, and used to screen 5×10⁵ clones of a human spleen λGT11cDNA library (Clontech) according to the manufacturer's instructions.Following hybridization, duplicating positives were rescreened with thesame probe until a pure positive phage plaque was obtained. Phage DNAwas recovered from positive plaques using standard methods (Sambrook J.et al (1989)). Purified phage DNA (100 μg) was digested with 200 unitsof Eco RI (New England Biolabs) in buffer 2 (New England Biolabs) for 16h at 37° C. Digestion products were fractionated on 1% agarose gelscontaining ethidium bromide (0.5 μg/ml) and cDNA inserts were gelpurified and ligated into the Eco RI site of pBluescript II SK- vectoras described above. Ligation products were transformed into E. colistrain XL-1 blue (recA1, endA1, gyrA96, thi-1, hsdR17, supE44, relA1,lac, {F' proAB, lacIqZDM15, Tn10 (tet^(r))] by electroporation aspreviously. Individual, ampicillin resistant bacterial colonies wereinoculated into L-Broth containing 100 μg/ml ampicillin and grown up for16 h at 37° C. Mini-prep DNA was prepared from 3 ml of overnight culturemedium as described above. Three μl aliquots of mini-prep DNA was thendigested with restriction enzyme Eco RI according to the manufacturers'instructions in a reaction volume of 15 μl. Reaction products wereanalysed on 1% agarose gels containing 0.5 μg/ml ethidium bromide.Mini-preps which contained cDNA inserts were subsequently sequencedusing Sequenase™ and T3 and T7 primers on an Applied Biosystems DNAsequencer.

One clone designated E1-C19, was shown by sequencing with the T7 primerto contain the putative 5' end of K5.5. CsCl gradient-purified DNA ofclone E1-C19 was subsequently resequenced with T3 and T7 primers andseveral internal sequencing primers based on the previous sequencingresults (primer sequences are shown in FIG. 2). The sequence of E1-C19insert cDNA is shown in FIG. 3.

(b) Northern Blot Analysis

Multiple tissue Northern blots were purchased from Clontech andhybridized to the 514 bp Hind III/Eco RI fragment of pTM(2-7)5.5according to the manufacturer's instructions. For other Northern blots,total RNA was prepared from cell lines and peripheral blood leukocytepopulations by the method of Chomczynski and Sacchi (1987). All of thecell lines used in this study were maintained in RPMI 1640 mediumcontaining 10% heat inactivated FCS and 50 μg/ml gentamycin (allpurchased from Gibco-BRL). Total peripheral blood mononuclear cells andpolymorphonuclear cells were purified by density gradient centrifugationon Ficoll (Pharmacia). Leukocytes were sorted by FACS using theappropriately labelled antibody on a FACS star (Becton Dickinson) toobtain pure populations (>90%) of B cells (CD20), T cells (CD4, CD8,CD45R0, CD45RA) and monocytes (CD14). Pulmonary macrophages and mixedlung leukocytes were prepared from resected human lung samples using themethod of Nicod et al (1989) (Separation of potent and poorly functionalhuman lung accessory cells based on autofluorescence. J. Leukocyte.Biol. 45 458).

5 μg of each RNA was electrophoresed in 1% agarose gels containing 2.2%(v/v) formaldehyde, transferred to nitrocellulose and probed with the ³²P-dCTP labelled 514 bp insert from TM(2-7)5.5 using standard Northernblot procedure (Sambrook et al (1989)). The results are shown in FIGS. 4to 6.

(c) Analysis of K5.5 Receptor mRNA Expression in Leukocytes and SomeHuman Cell Lines by Reverse Transcriptase PCR

10 μg of total RNA (in a volume of 10 μl) and 0.5 μl of 0.5 mg/mlsolution of oligodT₁₅ were heated at 70° C. for 10 min and then cooledon ice for 2 min, followed by addition of 4 μl of 5× 1st strand buffer,2 μl of 0.1 M DTT, 1 μl of 10 mM dNTPs and 1 μl Superscript™ for 1 h at37° C. All reagents for the reverse transcription (RT) reaction werefrom Gibco-BRL except oligodT₁₅ (Stratagene). Two μl aliquots of each RTreaction was then subjected to 40 cycles of PCR (2 min 95° C.; 2 min,55° C. and 2 min, 72° C.) in a 100 μl reaction mixture containing 100pmoles each of primers K5-5FLA and K5-5FLB. PCR reaction products (10μl) were analysed on 1% agarose gels as described above, for thepresence of a 1085 bp reaction product corresponding to the full codingsequence of K5.5. The results are shown in FIG. 7, wherein the samplesin the lanes indicated in FIG. 7 are as follows:

    ______________________________________                                        Lane        Sample                                                            ______________________________________                                         1          Molecular weight markers (1 kb ladder)                               2 PB T cells (IL-2 stimulated)                                                3 PB T cells                                                                  4 Jurkat                                                                      5 MOLT-4                                                                      6 PB B cells                                                                  7 PB B cells                                                                  8 pulmonary macrophages                                                       9 PB monocytes                                                               10 KU812                                                                      11 EOL-3                                                                      12 SW900 (lung epithelial cell line)                                          13 CCLu32 (lung fibroblast cell line)                                         14 LL24 (lung fibroblast cell line)                                           15 AALT.16 (aortic smooth muscle cell line)                                 ______________________________________                                    

(d) Expression of K5.5 cRNA in Xenopus Oocytes

CsCl-gradient purified pE1-C19 plasmid DNA (5 μg) was linearized usingrestriction enzyme Bam HI (New England Biolabs) in a 100 μl reactionvolume overnight at 37° C. Linearized plasmids were treated with 2 μl ofproteinase K (16.7 mg/ml Boehringer) for 30 min at 37° C. DNA wasextracted twice with phenol (0.1 M Tris-saturated pH 8.0) and once withchloroform. Glycogen (1 μl of 20 mg/ml stock solution) was added to theaqueous phase and linearized DNA was precipitated following addition of0.1 volume of 3 M sodium acetate pH 5.5 and 2.5 volumes of ethanol for 1h at -80° C. The DNA was recovered by centrifugation (14 000 rpm, 4° C.in a microfuge), washed with 70% ethanol and dissolved in RNase freewater at 250 ng/ml.

Capped cRNA transcripts were generated from 1 μg of Bam HI (New EnglandBiolabs) linearized DNA in a 100 μl reaction volume containing 20 μltranscription buffer (5×), 4 μl NTP mix (10 mM ATP, UTP and CTP, 3 mMGTP), 4 μl 0.75M DTT, 2.5 μl RNAsin, 0.5 μl GTP (10 mM), 4 μl CAP analog(10 mM m7G(5')ppp(5')G) and 2.5 μl of T7 or T3 RNA polymeraserespectively. All reagents used for the in vitro transcription reactionwere from Promega except CAP analog (Pharmacia). After 1.5 h at 37° C.,4 μl RQ1 DNase (Promega) was added and the reaction mixture wasincubated for a further 15 min at 37° C. The reaction mixture wasextracted twice with 0.1 M Tris-HCl pH 8.0 saturated phenol/chloroform(1:1 v/v) and once with chloroform. Glycogen (1 μl as above) was addedto the aqueous phase and cRNA was precipitated overnight at -20° C.after addition of 0.1 volume, 3 M sodium acetate pH 5.5 and 2.5 volumesethanol. cRNA was recovered by centrifugation (14 000 rpm, 4° C., 20 minin a microfuge), the pellet washed in 70% ethanol and resuspended insterile water at 1 μg/μl. An approximate estimate of the cRNAconcentration was obtained by running an aliquot of the resuspendedmaterial on a 1% agarose gel containing 2.2% (v/v) formaldehyde againstRNA markers of known concentration. Samples were stored at -80° C.before use.

Oocytes were harvested from adult female Xenopus laevis, by standardmethods (Bertrand et al., 1991). Oocytes were defollicullated byincubation in 0.2% (w/v) collagenase (Sigma) in 50 ml OR2 medium withoutCa2+ and without Mg2+ in a spinner flask under slow agitation for 2 h atroom temperature (OR2 medium is 82.5 mM NaCl, 2.5 mM KCl, 1 mM Na₂ HPO₄,15 mM HEPES, 2 mM CaCl₂, 1 mM MgCl₂ pH 7.6). Oocytes were rinsedcarefully with OR2 followed by MBS (modified Barth's saline: 88 mM NaCl,1 mM KCl, 0.33 mM Ca(NO₃)₂, 0.41 mM CaCl₂, 0.82 mM MgSO₄, 2.4 mM NaHCO₃,10 mM HEPES, pH 7.6) and allowed to recover for at least 1-2 h in MBSbefore selecting stage V-VI oocytes. Selected oocytes were incubated inMBS supplemented penicillin/streptomycin (100 units/ml) (Gibco-BRL)overnight at 18° C. before injection.

Oocytes were microinjected using an Inject+Matic air pump (Gabay) usingneedles made from Drummond calibrated 6 ml capillaries. cRNA (25 ng in50 nl) was injected into the cytoplasm. Oocytes were individuallytransferred to wells of a 96 well flat bottom culture dish and incubatedin MBS for 24-72 h.

Electrophysiological recordings were made 1-3 days after injection inoocytes superfused with OR2 medium at room temperature under voltageclamped conditions using two microelectrodes (1-2 MΩ, both filled with 3M KCl), the membrane potential being routinely clamped at -100 mV usinga Gene Clamp 500 instrument (Axon).

Test chemokines were purchased from PeptroTech or produced in-house atthe Glaxo Institute for Molecular Biology and resuspended at aconcentration of 1 μM in PBS. Fifty μl of each chemokine was applieddirectly onto voltage clamped oocytes and the current induced wasmonitored on a Tektronix 5113 dual-beam storage oscilloscope linked toan IBM-PC. Where multiple chemokines were tested on a single oocyte, arecovery time of 2 min was allowed between each application. The resultsare shown in FIG. 8A.

FIG. 8B shows the results of a similar analysis to that illustrated inFIG. 8A, but using different chemokines (apart from MIP-1α).

It can be seen that no significant electrophysiological response wasseen when using IL-8, in contrast with the result obtained for MIP-1α.

(e) HL-60 Cell Transfection and Ligand Binding Assay

Thirty μg human CC-CKR-3-pcDNA1neo, murine CC-CKR-3-pcDNA1neo, orpcDNA1neo were electroporated into 500 μl HL-60 cells (2×10⁷ cells/ml in0.15 M NaCl, 20 mM HEPES, pH 7.3) using a Bio Rad Geno Pulster (260volts, 960 μF, 0.4 cm gap cuvette). Cells were seeded into T-175 flaskscontaining 25 ml AIM-V serum-free media (GIBCO). On day 2 or 3 followingtransfection the cells were diluted in a total volume of 45 ml AIM-Vmedia containing 600 μg/ml G418, and on day 6, cells were furtherdiluted to 180 ml AIM-V media containing 600 μg/ml G418. On days 7-15post-transfection cells were maintained in AIM-V media (+G418) at adensity of 0.4-1.2×10⁶ cells/ml, and binding assays were performedduring this time. Equilibrium competition binding was carried out byincubating 5×10₅ cells in 100 μl binding buffer (1 mM CaCl₂, 5 mM MgCl₂,0.5% BSA, 50 mM HEPES, pH 7.2) , 0.34 nM [¹²⁵ I]radioligand, and0.5-2000 nM cold ligand in Millipore®-DV96-well filter plates. After 1.5h incubation at room temperature, cells were washed four times by vacuumfiltration with binding buffer containing 0.5 M NaCl. Fifty μl Optiphasescintillant (Wallac) were added to each well, and the radioactivity wasmeasured with a Wallac Microbeta Plate Reader. All binding data wasnormalized as the percentage of total binding. Total binding for a givenligand was defined as the radioactivity bound in the absence ofcompeting ligand to 5×10⁵ cells transfected with human CC-CKR-3 (range:1000-2500 cpm).

The results are shown in FIG. 9 which illustrates high affinity bindingof [¹²⁵ I]MIP-1α and [¹²⁵ I]RANTES to human and murine CC-CKR-3. HL-60cells were transfected with human CC-CKR-3 (), murine CC-CKR-3 (▪), oran empty vector (∘) and maintained in AIM-V media containing G418 for7-15 days. Equilibrium competition assays were performed as describedabove with [¹²⁵ I]MIP-1α (A) and [¹²⁵ I]RANTES (B). Each pointrepresents the mean ±S.D. of duplicate points from four (A) or three (B)separate experiments. Data were curve-fitted with GraFit 3.01 software(Leatherbarrow., R. J., GraFit Versions 3.01, Erithicus Softward Ltd.,Staines, UK (1992)) using the equation B/Bmax^(app) =1/(1+([L[/IC₅₀)),where B=cpm bound, Bmax^(app) =cpm bound in the absence of competingligand, L=competing ligand, and the IC₅₀ =[radioligand]+K_(d) (Cheng.,Y. and Prusoff., W. H. Biochem Pharmacol 22: 3099-3108 (1973)).

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 20                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - Arg Tyr Leu Ala Ile Val His Ala                                          1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Cys Xaa Asn Pro Xaa Xaa Tyr Xaa Xaa                                      1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "oligonucleotide"                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - GNTAYYTNGC NATHGTNCAY GC           - #                  - #                     22                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "oligonucleotide"                        - -     (iv) ANTI-SENSE: YES                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - AMNRCRTTAN ADNANNGGRT TNANRC          - #                  - #                  26                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 514 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 3..512                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - GG TAT CTG GCG ATA GTG CAC GCG GTG TTT TCC - # TTG AGG GCA AGG ACC            47                                                                          Tyr Leu Ala Ile Val His Ala Val Phe - #Ser Leu Arg Ala Arg Thr                  1             - #  5                - #  10                - #  15        - - TTG ACT TAT GGG GTC ATC ACC AGT TTG GCT AC - #A TGG TCA GTG GCT GTG           95                                                                       Leu Thr Tyr Gly Val Ile Thr Ser Leu Ala Th - #r Trp Ser Val Ala Val                            20 - #                 25 - #                 30              - - TTC GCC TCC CTT CCT GGC TTT CTG TTC AGC AC - #T TGT TAT ACT GAG CGC          143                                                                       Phe Ala Ser Leu Pro Gly Phe Leu Phe Ser Th - #r Cys Tyr Thr Glu Arg                        35     - #             40     - #             45                  - - AAC CAT ACC TAC TGC AAA ACC AAG TAC TCT CT - #C AAC TCC ACG ACG TGG          191                                                                       Asn His Thr Tyr Cys Lys Thr Lys Tyr Ser Le - #u Asn Ser Thr Thr Trp                    50         - #         55         - #         60                      - - AAG GTT CTC AGC TCC CTG GAA ANC AAC ATT CT - #C GGA TTG GTG ATC CCC          239                                                                       Lys Val Leu Ser Ser Leu Glu Xaa Asn Ile Le - #u Gly Leu Val Ile Pro                65             - #     70             - #     75                          - - TTA GGG ATC ATG CTG TTT TGC TAC TCC ATG AT - #C ATC AGG ACC TTG CAG          287                                                                       Leu Gly Ile Met Leu Phe Cys Tyr Ser Met Il - #e Ile Arg Thr Leu Gln            80                 - # 85                 - # 90                 - # 95       - - CAT TGT AAA AAT GAG AAG AAG AAC AAG GCG GT - #G AAG ATG ATC TTT GCC          335                                                                       His Cys Lys Asn Glu Lys Lys Asn Lys Ala Va - #l Lys Met Ile Phe Ala                           100  - #               105  - #               110              - - GTG GTG GTC CTC TTC CTT GGG TTC TGG ACA CC - #T TAC AAC ATA GTG CTC          383                                                                       Val Val Val Leu Phe Leu Gly Phe Trp Thr Pr - #o Tyr Asn Ile Val Leu                       115      - #           120      - #           125                  - - TTC CTA GAG ACC CTG GTG GAG CTA GAA GTC CT - #T CAG GAC TGC ACC TTT          431                                                                       Phe Leu Glu Thr Leu Val Glu Leu Glu Val Le - #u Gln Asp Cys Thr Phe                   130          - #       135          - #       140                      - - GAA AGA TAC TTG GAC TAT GCC AGC CAG GCC AC - #A GAA ACT CTG GCT TTT          479                                                                       Glu Arg Tyr Leu Asp Tyr Ala Ser Gln Ala Th - #r Glu Thr Leu Ala Phe               145              - #   150              - #   155                          - - GTT CAC TGC TGC CTC AAT CCC CTC CTC TAC GC - #C GT                -     #       514                                                                    Val His Cys Cys Leu Asn Pro Leu Leu Tyr Al - #a                               160                 1 - #65                 1 - #70                            - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 170 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - Tyr Leu Ala Ile Val His Ala Val Phe Ser Le - #u Arg Ala Arg Thr Leu        1               5 - #                 10 - #                 15              - - Thr Tyr Gly Val Ile Thr Ser Leu Ala Thr Tr - #p Ser Val Ala Val Phe                   20     - #             25     - #             30                  - - Ala Ser Leu Pro Gly Phe Leu Phe Ser Thr Cy - #s Tyr Thr Glu Arg Asn               35         - #         40         - #         45                      - - His Thr Tyr Cys Lys Thr Lys Tyr Ser Leu As - #n Ser Thr Thr Trp Lys           50             - #     55             - #     60                          - - Val Leu Ser Ser Leu Glu Xaa Asn Ile Leu Gl - #y Leu Val Ile Pro Leu       65                 - # 70                 - # 75                 - # 80       - - Gly Ile Met Leu Phe Cys Tyr Ser Met Ile Il - #e Arg Thr Leu Gln His                       85 - #                 90 - #                 95              - - Cys Lys Asn Glu Lys Lys Asn Lys Ala Val Ly - #s Met Ile Phe Ala Val                  100      - #           105      - #           110                  - - Val Val Leu Phe Leu Gly Phe Trp Thr Pro Ty - #r Asn Ile Val Leu Phe              115          - #       120          - #       125                      - - Leu Glu Thr Leu Val Glu Leu Glu Val Leu Gl - #n Asp Cys Thr Phe Glu          130              - #   135              - #   140                          - - Arg Tyr Leu Asp Tyr Ala Ser Gln Ala Thr Gl - #u Thr Leu Ala Phe Val      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - His Cys Cys Leu Asn Pro Leu Leu Tyr Ala                                                  165  - #               170                                     - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "primer"                                 - -     (iv) ANTI-SENSE: YES                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - AGAGTACTTG GTTTTGCAGT AG           - #                  - #                     22                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "primer"                                 - -     (iv) ANTI-SENSE: YES                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - GCAGCAGTGA ACAAAAGCCA G           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "primer"                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - CATAGTGCTC TTCCTAGAGA C           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "primer"                                 - -     (iv) ANTI-SENSE: YES                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - GGTTGAGCAG GTACACATCA G           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "primer"                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - CAATACTGTG GGCTCCTCC             - #                  - #                      - # 19                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "primer"                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - GCTCAGGTCC ATGACTG             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "primer"                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - CTCATGAGCA TTGATAG             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "primer"                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - CTGAGCGCAA CCATACC             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "primer"                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - GCTAGAAGTC CTTCAGG             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "primer"                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - GGATCATGAT CTTCATG             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "primer"                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - - AAATGAAACC CCACGGATAT AGCAG          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "primer"                                 - -     (iv) ANTI-SENSE: YES                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                              - - TCCTACAGAG CATCATGAAG ATC           - #                  - #                    23                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1607 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 183..1262                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                              - - CGGGGGTTTT GATCTTCTTC CCCTTCTTTT CTTCCCCTTC TTCTTTCCTT CC -             #TCCCTCCC     60                                                                 - - TCTCTCATTT CCCTTCTCCT TCTCCCTCAG TCTCCACATT CAACATTGAC AA -            #GTCCATTC    120                                                                 - - AGAAAAGCAA GCTGCTTCTG GTTGGGCCCA GACCTGCCTT GAGGAGCCTG TA -            #GAGTTAAA    180                                                                 - - AA ATG AAC CCC ACG GAT ATA GCA GAT ACC ACC - # CTC GAT GAA AGC ATA           227                                                                         Met Asn Pro Thr Asp Ile Ala Asp Thr - #Thr Leu Asp Glu Ser Ile                  1             - #  5                - #  10                - #  15        - - TAC AGC AAT TAC TAT CTG TAT GAA AGT ATC CC - #C AAG CCT TGC ACC AAA          275                                                                       Tyr Ser Asn Tyr Tyr Leu Tyr Glu Ser Ile Pr - #o Lys Pro Cys Thr Lys                            20 - #                 25 - #                 30              - - GAA GGC ATC AAG GCA TTT GGG GAG CTC TTC CT - #G CCC CCA CTG TAT TCC          323                                                                       Glu Gly Ile Lys Ala Phe Gly Glu Leu Phe Le - #u Pro Pro Leu Tyr Ser                        35     - #             40     - #             45                  - - TTG GTT TTT GTA TTT GGT CTG CTT GGA AAT TC - #T GTG GTG GTT CTG GTC          371                                                                       Leu Val Phe Val Phe Gly Leu Leu Gly Asn Se - #r Val Val Val Leu Val                    50         - #         55         - #         60                      - - CTG TTC AAA TAC AAG CGG CTC AGG TCC ATG AC - #T GAT GTG TAC CTG CTC          419                                                                       Leu Phe Lys Tyr Lys Arg Leu Arg Ser Met Th - #r Asp Val Tyr Leu Leu                65             - #     70             - #     75                          - - AAC CTT GCC ATC TCG GAT CTG CTC TTC GTG TT - #T TCC CTC CCT TTT TGG          467                                                                       Asn Leu Ala Ile Ser Asp Leu Leu Phe Val Ph - #e Ser Leu Pro Phe Trp            80                 - # 85                 - # 90                 - # 95       - - GGC TAC TAT GCA GCA GAC CAG TGG GTT TTT GG - #G CTA GGT CTG TGC AAG          515                                                                       Gly Tyr Tyr Ala Ala Asp Gln Trp Val Phe Gl - #y Leu Gly Leu Cys Lys                           100  - #               105  - #               110              - - ATG ATT TCC TGG ATG TAC TTG GTG GGC TTT TA - #C AGT GGC ATA TTC TTT          563                                                                       Met Ile Ser Trp Met Tyr Leu Val Gly Phe Ty - #r Ser Gly Ile Phe Phe                       115      - #           120      - #           125                  - - GTC ATG CTC ATG AGC ATT GAT AGA TAC CTG GC - #G ATA GTG CAC GCG GTG          611                                                                       Val Met Leu Met Ser Ile Asp Arg Tyr Leu Al - #a Ile Val His Ala Val                   130          - #       135          - #       140                      - - TTT TCC TTG AGG GCA AGG ACC TTG ACT TAT GG - #G GTC ATC ACC AGT TTG          659                                                                       Phe Ser Leu Arg Ala Arg Thr Leu Thr Tyr Gl - #y Val Ile Thr Ser Leu               145              - #   150              - #   155                          - - GCT ACA TGG TCA GTG GCT GTG TTC GCC TCC CT - #T CCT GGC TTT CTG TTC          707                                                                       Ala Thr Trp Ser Val Ala Val Phe Ala Ser Le - #u Pro Gly Phe Leu Phe           160                 1 - #65                 1 - #70                 1 -      #75                                                                              - - AGC ACT TGT TAT ACT GAG CGC AAC CAT ACC TA - #C TGC AAA ACC AAG        TAC      755                                                                    Ser Thr Cys Tyr Thr Glu Arg Asn His Thr Ty - #r Cys Lys Thr Lys Tyr                          180  - #               185  - #               190              - - TCT CTC AAC TCC ACG ACG TGG AAG GTT CTC AG - #C TCC CTG GAA ATC AAC          803                                                                       Ser Leu Asn Ser Thr Thr Trp Lys Val Leu Se - #r Ser Leu Glu Ile Asn                       195      - #           200      - #           205                  - - ATT CTC GGA TTG GTG ATC CCC TTA GGG ATC AT - #G CTG TTT TGC TAC TCC          851                                                                       Ile Leu Gly Leu Val Ile Pro Leu Gly Ile Me - #t Leu Phe Cys Tyr Ser                   210          - #       215          - #       220                      - - ATG ATC ATC AGG ACC TTG CAG CAT TGT AAA AA - #T GAG AAG AAG AAC AAG          899                                                                       Met Ile Ile Arg Thr Leu Gln His Cys Lys As - #n Glu Lys Lys Asn Lys               225              - #   230              - #   235                          - - GCG GTG AAG ATG ATC TTT GCC GTG GTG GTC CT - #C TTC CTT GGG TTC TGG          947                                                                       Ala Val Lys Met Ile Phe Ala Val Val Val Le - #u Phe Leu Gly Phe Trp           240                 2 - #45                 2 - #50                 2 -      #55                                                                              - - ACA CCT TAC AAC ATA GTG CTC TTC CTA GAG AC - #C CTG GTG GAG CTA        GAA      995                                                                    Thr Pro Tyr Asn Ile Val Leu Phe Leu Glu Th - #r Leu Val Glu Leu Glu                          260  - #               265  - #               270              - - GTC CTT CAG GAC TGC ACC TTT GAA AGA TAC TT - #G GAC TAT GCC ATC CAG         1043                                                                       Val Leu Gln Asp Cys Thr Phe Glu Arg Tyr Le - #u Asp Tyr Ala Ile Gln                       275      - #           280      - #           285                  - - GCC ACA GAA ACT CTG GCT TTT GTT CAC TGC TG - #C CTT AAT CCC ATC ATC         1091                                                                       Ala Thr Glu Thr Leu Ala Phe Val His Cys Cy - #s Leu Asn Pro Ile Ile                   290          - #       295          - #       300                      - - TAC TTT TTT CTG GGG GAG AAA TTT CGC AAG TA - #C ATC CTA CAG CTC TTC         1139                                                                       Tyr Phe Phe Leu Gly Glu Lys Phe Arg Lys Ty - #r Ile Leu Gln Leu Phe               305              - #   310              - #   315                          - - AAA ACC TGC AGG GGC CTT TTT GTG CTC TGC CA - #A TAC TGT GGG CTC CTC         1187                                                                       Lys Thr Cys Arg Gly Leu Phe Val Leu Cys Gl - #n Tyr Cys Gly Leu Leu           320                 3 - #25                 3 - #30                 3 -      #35                                                                              - - CAA ATT TAC TCT GCT GAC ACC CCC AGC TCA TC - #T TAC ACG CAG TCC        ACC     1235                                                                    Gln Ile Tyr Ser Ala Asp Thr Pro Ser Ser Se - #r Tyr Thr Gln Ser Thr                          340  - #               345  - #               350              - - ATG GAT CAT GAT CTC CAT GAT GCT CTG TAGAAAAAT - #G AAATGGTGAA               1282                                                                       Met Asp His Asp Leu His Asp Ala Leu                                                       355      - #           360                                         - - ATGCAGAGTC AATGAACTTT TCCACATTCA GAGCTTACTT TAAAATTGGT AT -             #TTTTAGGT   1342                                                                 - - AAGAGATCCC TGAGCCAGTG GTCAGGAGGA AAGGCTTACA CCCACAGGTG GG -            #AAAGACAG   1402                                                                 - - GTTCTCATCC CTGCAGGNAG GTTTTTCTTC TCCCCACTTA GANAAAGTNC CA -            #GGCCTGGA   1462                                                                 - - AGGGGTCCAA CCCNGGGTTG AGGATCCTTC CCCCAAACCC AGGGTTTGGC CT -            #GGAGGATT   1522                                                                 - - AATNCAAAAN NTTTNTTGAA ACTCTTGAAN ANGTTGNGNT AAGTTTNGGG GG -            #GTTNTTTT   1582                                                                 - - GAAGGNAAGT TTTTCCCTTC TTNCC          - #                  - #                 1607                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 360 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                              - - Met Asn Pro Thr Asp Ile Ala Asp Thr Thr Le - #u Asp Glu Ser Ile Tyr        1               5 - #                 10 - #                 15              - - Ser Asn Tyr Tyr Leu Tyr Glu Ser Ile Pro Ly - #s Pro Cys Thr Lys Glu                   20     - #             25     - #             30                  - - Gly Ile Lys Ala Phe Gly Glu Leu Phe Leu Pr - #o Pro Leu Tyr Ser Leu               35         - #         40         - #         45                      - - Val Phe Val Phe Gly Leu Leu Gly Asn Ser Va - #l Val Val Leu Val Leu           50             - #     55             - #     60                          - - Phe Lys Tyr Lys Arg Leu Arg Ser Met Thr As - #p Val Tyr Leu Leu Asn       65                 - # 70                 - # 75                 - # 80       - - Leu Ala Ile Ser Asp Leu Leu Phe Val Phe Se - #r Leu Pro Phe Trp Gly                       85 - #                 90 - #                 95              - - Tyr Tyr Ala Ala Asp Gln Trp Val Phe Gly Le - #u Gly Leu Cys Lys Met                  100      - #           105      - #           110                  - - Ile Ser Trp Met Tyr Leu Val Gly Phe Tyr Se - #r Gly Ile Phe Phe Val              115          - #       120          - #       125                      - - Met Leu Met Ser Ile Asp Arg Tyr Leu Ala Il - #e Val His Ala Val Phe          130              - #   135              - #   140                          - - Ser Leu Arg Ala Arg Thr Leu Thr Tyr Gly Va - #l Ile Thr Ser Leu Ala      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Thr Trp Ser Val Ala Val Phe Ala Ser Leu Pr - #o Gly Phe Leu Phe        Ser                                                                                             165  - #               170  - #               175             - - Thr Cys Tyr Thr Glu Arg Asn His Thr Tyr Cy - #s Lys Thr Lys Tyr Ser                  180      - #           185      - #           190                  - - Leu Asn Ser Thr Thr Trp Lys Val Leu Ser Se - #r Leu Glu Ile Asn Ile              195          - #       200          - #       205                      - - Leu Gly Leu Val Ile Pro Leu Gly Ile Met Le - #u Phe Cys Tyr Ser Met          210              - #   215              - #   220                          - - Ile Ile Arg Thr Leu Gln His Cys Lys Asn Gl - #u Lys Lys Asn Lys Ala      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Val Lys Met Ile Phe Ala Val Val Val Leu Ph - #e Leu Gly Phe Trp        Thr                                                                                             245  - #               250  - #               255             - - Pro Tyr Asn Ile Val Leu Phe Leu Glu Thr Le - #u Val Glu Leu Glu Val                  260      - #           265      - #           270                  - - Leu Gln Asp Cys Thr Phe Glu Arg Tyr Leu As - #p Tyr Ala Ile Gln Ala              275          - #       280          - #       285                      - - Thr Glu Thr Leu Ala Phe Val His Cys Cys Le - #u Asn Pro Ile Ile Tyr          290              - #   295              - #   300                          - - Phe Phe Leu Gly Glu Lys Phe Arg Lys Tyr Il - #e Leu Gln Leu Phe Lys      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Thr Cys Arg Gly Leu Phe Val Leu Cys Gln Ty - #r Cys Gly Leu Leu        Gln                                                                                             325  - #               330  - #               335             - - Ile Tyr Ser Ala Asp Thr Pro Ser Ser Ser Ty - #r Thr Gln Ser Thr Met                  340      - #           345      - #           350                  - - Asp His Asp Leu His Asp Ala Leu                                                  355          - #       360                                           __________________________________________________________________________

What is claimed is:
 1. A vector comprising a nucleic acid molecule thatcodes for a polypeptide wherein said polypeptide comprises the aminoacid sequence shown in SEQ ID NO:20.
 2. A host comprising a vector thatcomprises a nucleic acid molecule that codes for a polypeptide whereinsaid polypeptide comprises the amino acid sequence shown in SEQ IDNO:20.
 3. An isolated nucleic acid that codes for a polypeptide whereinsaid polypeptide has the amino acid sequence shown in SEQ ID NO:20. 4.An isolated nucleic acid that has the nucleotide sequence shown in SEQID NO:19.
 5. An isolated nucleic acid that comprises nucleotides183-1262 of the nucleotide sequence shown in SEQ ID NO:19.
 6. Anisolated nucleic acid molecule that codes for a polypeptide wherein saidpolypeptide comprises the amino acid sequence shown in SEQ ID NO:6. 7.An isolated nucleic acid molecule that codes for a polypeptide whereinsaid polypeptide has the amino acid sequence shown in SEQ ID NO:6.
 8. Anisolated nucleic acid molecule that comprises the nucleotide sequenceshown in SEQ ID NO:5.
 9. An isolated nucleic acid molecule that has thenucleotide sequence shown in SEQ ID NO:5.
 10. An isolated nucleic acidmolecule that codes for a polypeptide wherein said polypeptide comprisesthe amino acid sequence shown in SEQ ID NO:20.
 11. A polypeptideaccording to claim 2 which has the amino acid sequence shown in FIG. 3(SEQ ID NO:20).
 12. An isolated polypeptide that comprises the aminoacid sequence shown in SEQ ID NO:20.
 13. A method for producing thepolypeptide according to claim 12 comprising incubating a hostcomprising a vector that comprises a nucleic acid molecule that codesfor the polypeptide according to claim 12 under conditions causingexpression of said polypeptide and then purifying said polypeptide. 14.An isolated polypeptide that comprises the amino acid sequence shown inSEQ ID NO:6.
 15. An isolated polypeptide according to claim 14 that hasthe amino acid sequence shown in SEQ ID NO:6.
 16. A method ofidentifying an agent which affects the interaction of a polypeptidecomprising the amino acid sequence shown in SEQ ID NO:20 with RANTES,MIP-1α or MCP-1 wherein said method comprises assaying the binding ofsaid agent to said polypeptide.
 17. The method according to claim 16wherein said method of assaying comprises an electrophysiologicaltechnique.
 18. The method according to claim 16 wherein said method ofassaying comprises use of a ligand with a detectable label.